Research on new antibiotics is critically needed at this point in history as many microorganisms have become resistant to the currently available antibiotic drugs. One area of current interest is the pleuromutilin class of antibacterial agents. Retapamulin, a semisynthetic derivative of pleuromutilin, was approved in 2007 to treat skin infections, including Staph infections. Research suggests that resistance to retapamulin is likely to develop slowly, which suggests that this class of drugs will be a useful addition to current antibiotics. The focus of our research is to discover how pleuromutilin, the fungal natural product precursor to retapamulin, is produced by the fungus Clitopilus passeckerianus. Within the project period, we aim to discover the biosynthetic gene cluster responsible for the formation of pleuromutilin and to characterize the specific diterpene synthase and cytochrome P450 enzymes responsible for forming the pleuromutilin core. Structurally, pleuromutilin is a member of the diterpene family of isoprenoid natural products. Full genome sequencing utilizing next generation sequencing technologies (Illumina sequencing) will provide sequence of sufficient quality to identify the putative gene cluster using a bioinformatics approach, which will be verified by biochemical characterization of the recombinant enzymes. Knowledge of the biosynthetic genes should eventually allow us to increase the efficiency of pleuromutilin production, providing the starting material for the semi-synthesis of promising antibacterial agents. Additionally, characterization of the novel diterpene synthase that forms the pleuromutilin skeleton will add to our knowledge of these diverse enzymes that form the structural scaffolds to many bioactive compounds. PUBLIC HEALTH RELEVANCE: The pleuromutilin class of antibiotics, derived from the fungal natural product pleuromutilin, has recently been approved for the treatment of human bacterial infections, including those resistant to other therapies. An understanding of the fungal genes necessary to form pleuromutilin will allow for rational approaches to improve the production of the common core structure of these antibiotics and will provide a greater understanding of how nature forms these important compounds.